Project Summary Myelodysplastic syndromes (MDS) are hematopoietic disorders characterized by ineffective hematopoiesis, peripheral cytopenias, and a propensity for progression to acute myeloid leukemia (AML). MDS remains incurable by existing nontransplant therapy, which is the only option for MDS patients over 60 years old. An increasing number of clonal-analysis studies provide direct evidence that, in MDS, cells of the entire bone marrow are clonally derived from a single hematopoietic stem cell (HSC) or early myeloid progenitor. These aberrant MDS hematopoietic stem and progenitor cells (HSPCs) reportedly resist therapy and expand causing relapse. Thus, improved understanding of mechanisms regulating MDS HSPCs maintenance could foster development of therapies targeting MDS HSPCs. The DNA demethylation enzyme Tet methylcytosine dioxygenase 2 (TET2) reportedly protects normal HSCs from transformation into disease-initiating clones in hematological malignancies. We recently found that protein levels of SIRT1, a deacetylase that contributes to normal HSC maintenance under stress conditions, significantly decreased in MDS CD34+ cells, a population highly enriched for MDS HSPCs. Using loss-of- function and mutagenesis studies, we identified a novel mechanism that SIRT1-deficiency induces TET2 hyperacetylation, leading to TET2 dysfunction in MDS cells. We also found that SIRT1 activation blocked MDS cell proliferation in a TET2-dependent manner. Importantly, our preliminary studies also show that TET2 acetylation levels increase in human MDS specimens expressing below normal SIRT1 protein levels. Based on these findings, we hypothesize that, in the absence of TET2 mutations, SIRT1-deficiency induces TET2 dysfunction due to unregulated hyperacetylation, enabling MDS HSPCs maintenance, and accordingly, that SIRT1-induced TET2 deacetylation could ablate MDS HSPCs. To test our hypothesis, we will: 1) determine the pathogenic roles of SIRT1 and TET2 in MDS maintenance using genetic mouse models; 2) define upstream and downstream factors of the SIRT1/TET2 axis in MDS cells; and 3) determine whether SIRT1 activation alone or in combination with a hypomethylating agent that is currently first-line treatment for older, high risk MDS patients can ablate MDS HSPCs. We expect that our studies will uncover functional interaction between SIRT1 and TET2 and reveal how both factors govern MDS HSPC growth and self-renewal. These studies will close the knowledge gap relevant to how MDS-initiating clones acquire a growth advantage during MDS development and may identify more effective therapeutic strategy for ablating MDS disease- propagating cells by targeting SIRT1.